A HEURISTIC APPROACH TO THE ANALYSIS OF ENZYMATIC CATALYSIS - REACTION OF HA-AMINOADIPOYL)-L-CYSTEINYL-D-ALPHA-AMINOBUTYRATE AND -(L-ALPHA-AMINOADIPOYL)-L-CYSTEINYL-D-ALLYLGLYCINE CATALYZED BY ISOPENICILLIN-N SYNTHASE ISOZYMES

Isopenicillin N synthase (IPNS) catalyzes the oxidative cyclization of delta-(L-alpha-aminoadipoyl)L-cysteinyl-D-valine to isopenicillin N. It is proposed that the multiple products produced from certain substr ate analogues result from pathway branching after formation of a ferry l oxene intermediate. We have been interested in ascertaining the reas ons for multiple product formation. One possibility is that the produc ts are predisposed toward formation once the beta-lactam ring and the ferryl, oxene are produced. Alternately, the products may be persuaded into being by the enzyme restricting conformations such that otherwis e less favorable chemistry can take place. With the existing descripti on of the IPNS catalytic cycle, this fundamental question has not been answerable. We describe here the application of a heuristic method to resolve this key issue. It was reasoned that by comparing the ratios of products formed by a set of perturbed IPNS variants it might be pos sible to generate qualitative information about the relative magnitude of certain activation parameters. If certain product ratios are affec ted but others are not, then it should be possible to say which steps in the reaction are dictated merely by chemical fundamentals and which steps are directly effected by the enzyme. In this paper we report th e high-level expression, purification, and characterization of four IP NS isozymes. Comparison of the product ratios obtained on incubation o f unnatural substrate analogues with four IPNS isozymes corresponding to perturbed active site variants shows substantial variation in some cases and little in others. Interpretation of the results obtained wit h ha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate (ACAB) allows con clusions to be drawn regarding the role of the enzyme in restricting a vailable conformations of the natural substrate to disfavor certain ot herwise chemically favorable pathways and hence products. The results obtained with -(L-alpha-aminoadipoyl)-L-cysteinyl-D-allylglycine while rather more complex, substantiate the conclusions drawn from the ACAB data. A major conclusion is that, in the oxidation of ACV, IPNS is a negative catalyst of cepham formation but a positive catalyst of penam formation.